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E.E. Arnett, T.M. Seeberger, A.V. Crum, J.I. Clark; In vivo Fluorescence Imaging of Mouse Lenses . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2909.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To develop a simple, in vivo imaging for slit lamp observation in mice. Introduction: Enhanced Green Fluorescent Protein (EGFP, from Clonetech Laboratories, Inc.) is a common label for protein expression in vivo. Mice expressing an EGFP– labeled expression product were examined using emission and barrier filters with selected wavelengths to observe spatial distributions in living animal lenses. Methods: Unanesthetized mice were examined using a slit lamp. Eyes were dilated with a drop of 0.5% tropicamide, 5% phenylephrine solution and held in front of a slit lamp (Nikon FS–2). Slit lamp examinations, at 40X magnification, were recorded using an integrated digital video camera. Three absorption filters were utilized in the examination. An excitation filter allows a narrow range of wavelengths (465–498nm) to excite EGFP (max: 488) in the lens. An emission filter (515–560nm), was used with the excitation filter, for transmission of EGFP emission (max: 510nm). For visualization of opacity without interference of EGFP emission, a barrier filter prevented EGFP transmission (475–560nm). Frames were selected from video recordings, using Adobe® Premier®, saved in .tif file format, and then cropped and oriented using Adobe® Photoshop®. Results: Anterior views were used to observed product expression in the lens. Slit lamp views allowed detailed observation of the lens. Opacities in the subnuclear cortex and the nucleus of the lens were apparent in the slit view without filters. Using the excitation filter, expression of the transgene was observed mostly in the subcortical region of the lens. With excitation and emission filters, EGFP expression was observed in the subcortical region of the transgenic mice. The slit view with the EGFP blocking filter confirmed that there was opacity that corresponded with position and pattern of product expression. Conclusions: Slit lamp fluorescence imaging is a simple and rapid method for the evaluation of EGFP expression products associated with opacity in the living mouse lens. EGFP excitation and emission filters were necessary for distinguishing the expression of EGFP–labeled product from normal lens constituents seen without filters. The EGFP blocking filter was necessary to distinguish total expression from aggregated EGFP labeled product.
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